Figure 1.
Experimental setup with PDMS devices.
(A) Schematic representations (axonometric view) of the PDMS microdevice (left panel), of a spheroid that has just been placed in the PDMS microdevice (middle panel) and after 6 days of culture (right panel). (B) Transmitted-light images (top view) of a PDMS microdevice (left panel) and of a spheroid at day 0, day 3 and day 6 of growth within the microdevice (scale bar, 200 µm). (C) Schematic representation of how control MCTS (grown in non-confined conditions) and mechanically confined MCTS (grown in the PDMS microdevice) were oriented for cryosectioning. (D) Schematic representations of sections of control and confined MCTS. The dashed lines indicate the PDMS walls. The peripheral area corresponds to a 60 µm-width region along the surface of control MCTS or along the body walls of confined MCTS. The central area corresponds to a 60 µm-width region 120 µm away from the surface in control MCTS and from the PDMS walls in the body of confined MCTS.
Figure 2.
MCTS grown in PDMS microdevices are mechanically stressed.
(A) Cell density in the peripheral (P) and central (C) areas of control MCTS (48 ROI analyzed from 15 MCTS obtained in 3 independent experiments) and in the body (56 ROI analyzed), and tips (39 ROI analyzed) of 6 confined MCTS from 3 independent experiments. The bars correspond to the mean ± SEM. (B) Transmitted-light images of an MCTS were acquired at the indicated times after removal from the PDMS microdevice in which it was cultured for 6 days. Images are extracted from the time-lapse experiment (Zeiss Axiovert microscope) shown in Movie S1. (C) Change in roundness values of 5 MCTS (from 5 independent experiments) over time after removal from the PDMS microdevice in which they were cultured for 6 days. The roundness values were calculated as 4x(area)/(πx(major axis)2) at each time point up to 24 hours after removal. A rapid swelling of the MCTS is observed just after the removal and then roundness increases more slowly.
Figure 3.
Proliferation, hypoxia and apoptosis within MCTS grown in PDMS microdevices.
(A) Transmitted light images of control and confined MCTS grown in a PDMS microdevice for 6 days. (B) Detection by immunofluorescence of Ki67 staining (marker of proliferative cells). Mean fluorescence intensity of 10 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (6 days in the PDMS microdevice; from 3 independent experiments). (C) Detection by immunofluorescence of Cyclin A staining. Mean fluorescence intensity of 6 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (from 3 independent experiments). (D) Detection by immunofluorescence of hypoxia (pimonidazole; in green). Mean fluorescence intensity of 8 cryosections from 8 different control MCTS (from 3 independent experiments) and of 8 cryosections from 6 mechanically confined MCTS (from 4 independent experiments). The grey circle indicates the MCTS margins. (E) Cleaved PARP staining (apoptosis marker). Mean fluorescence intensity of 6 cryosections from 6 control MCTS (two independent experiments) and of 6 cryosections from 6 mechanically confined MCTS (6 days in the PDMS device; 4 independent experiments). c-PARP, cleaved PARP. The grey circle indicates the MCTS margins. (A–E) The dashed lines indicate the PDMS walls. (A–D) The color scale indicates the fluorescence intensity (scale bar, 100 µm.). Nuclei are stained by DAPI (blue).
Figure 4.
Mechanically confined growth impairs the regionalization of mitotic cells in MCTS.
(A) Transmitted light images of a control MCTS and of a MCTS grown in a PDMS microdevice for 6 days. (B) Upper panels: Detection by immunofluorescence of mitotic cells (anti-phosphorylated Histone H3 antibody, pH3; in green) in cryosections of a control MCTS and a mechanically confined MCTS. The orientation of mechanically confined MCTS cryosections is parallel to the bottom of the channel (middle, see also Fig 1C) and perpendicular to the bottom of the channel (right, see also Fig 1C). Nuclei are stained with DAPI (blue). Lower panels: mean fluorescence intensity of pH3 staining in 8 cryosections from 6 control MCTS (3 independent experiments), 11 parallel cryosections from 11 mechanically confined MCTS (4 independent experiments) and 8 perpendicular cryosections from 6 mechanically confined MCTS (3 independent experiments). Dashed lines represent the walls of the PDMS channel. White lines indicate the width of the area where mitotic cells are localized (scale bar, 100 µm). (C) Percentages of mitotic cells (pH3-positive cells) in the peripheral (P) and the central (C) areas of control MCTS (n = 14 areas analyzed, from 7 MCTS from 3 experiments) and in the peripheral (P) and central (C) areas and the tips (Tips) of confined MCTS (n = 29 areas analyzed, from 12 MCTS from 6 experiments). The bars correspond to the mean ± SEM.
Figure 5.
Growth-associated external mechanical stress leads to accumulation of cells arrested in mitosis.
(A) Upper panel: Immunodetection of EdU incorporation (green) and mitotic cells (pH3-positive, red) in a cryosection from MCTS grown in PDMS microdevices for 6 days. Nuclei are stained using DAPI (blue). Lower panel: High-contrast image of the immunodetection of EdU incorporation (white) (scale bar, 100 µm). (B) Analysis of EdU incorporation in mitotic cells. Images correspond to magnifications of the regions indicated by the white squares in A from the tip (top panels) and the body (bottom panels) of a mechanically confined MCTS. The white arrow indicates a pH3- positive cell that is not EdU-positive. This cell is next to a pH3-positive/EdU-positive cell. (C) Percentage of pH3-positive/EdU-negative cells in the body (589 mitotic cells from 48 cryosections) and in the tips (331 mitotic cells from 95 cryosections) of mechanically confined MCTS (20 MCTS from 4 independent experiments) and in control (CTL) MCTS (358 mitotic cells from 34 cryosections from 15 MCTS from 4 independent experiment). Bars correspond to the mean ± SEM. (D) Map showing the localization of pH3-positive/EdU-negative cells in 8 cryosections from 8 mechanically confined MCTS from 4 independent experiments. The white line represents the outline of the MCTS and the red dots the localization of the pH3-positive/EdU-negative cells. The dashed lines indicate the microdevice PDMS walls.
Figure 6.
Mechanically confined MCTS show bipolar spindle defects.
(A) Maximal projection of two mitotic cells from two z-stacks of images of a cryosection from a mechanically confined MCTS (6 days in the PDMS microdevice) incubated with an anti-γTubulin antibody (green). Nuclei were stained with DAPI (blue) (scale bar, 5 µm). (B) Distribution (percentage) of mitotic cells as a function of the number of spindle poles in control MCTS (CTL, 37 mitotic cells analyzed from 10 MCTS from 3 independent experiment) and in the body of mechanically confined MCTS (Confined, 66 mitotic cells analyzed from 8 MCTS from 4 independent experiment). (C) Distribution of the pole-to-pole distance (in µm) in bipolar mitotic cells from the control (CTL) and mechanically confined (Confined) MCTS analyzed in (B). The lines correspond to the mean ± SD.
Figure 7.
Mechanical confinement does not impair mitotic cell rounding within MCTS.
(A) Cryosections of a control MCTS and a mechanically confined MTCS (6 days in the PDMS device), stained for DNA (blue) and E-Cadherin (green) (scale bar, 10 µm). The cell outlines are drawn manually (green dashed line) to extract the area and the circularity of cells. (B) Area values of interphase cells (blue) and mitotic cells (orange) in control MCTS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). Lines correspond to the mean ± SD. (C) Circularity values of interphase cells (blue) and mitotic cells (orange) in control MTCS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). The error bars represent the mean ± SD. For control MCTS, 167 interphase cells and 175 mitotic cells were analyzed from 8 MCTS from 2 experiments. For confined MCTS, 307 interphase cells and 125 mitotic cells were analyzed in the body, and 146 interphase cells and 91 mitotic cells were analyzed in the tip, both from 10 MCTS from 4 experiments. (D) Cryosections of a mechanically confined MCTS stained for DNA (blue), E-Cadherin (grey), EdU (green) and pH3 (red) (scale bar, 10 µm). (E) Area (left panel) and circularity (right panel) values of pH3-positive mitotic cells in the body of mechanically confined MCTS relative to EdU incorporation (EdU+/pH3+: 79 cells analyzed, EdU−/pH3+: 43 cells analyzed, from 3 MCTS from 2 independent experiments). The lines correspond to the mean ± SD.